Allograft transplantation provokes miRNA 297–669 cluster that reduces T regulatory cell generation through targeting TGF-β2

Abstract

Abstract T regulatory cells (Tregs) are anti-inflammatory cells capable of quelling the harshest immune responses. Accordingly, Treg therapy is rapidly becoming a viable treatment option for patients stricken with autoimmune diseases or those in need of an organ transplant. In a model of allotransplantation, via microarray, our work identified miRNA cluster 297–669 is induced in CD4+ cells in the lymph nodes draining the allograft in C57BL/6 mice given a C3H skin transplant. One member of this cluster, miR 466a-3p, is induced in a co-culture of lymph node cells stimulated with alloantigen in vitro. We have shown that this miRNA specifically targets transforming growth factor beta 2 (TGF-β2). The reduction in TGF-β2 levels in draining lymph node CD4+ T cells caused a resultant decrease in the generation of FoxP3+ Tregs, both within the graft and in the draining lymph node. Furthermore, blocking the effect of this miRNA using a specific locked nucleic acid (LNA) reversed this effect. Generation of Tregs using transforming growth factor beta1 (TGF-β1), interleukin-2 and CD3/CD28 is the most common method of induction. Here we show that replacing TGF-β1 with TGF-β2 has no detrimental effect on Treg induction. Indeed, Tregs induced with TGF-β2 have equal in vivo suppressive capability as TGF-β1 Tregs in a skin graft model when adoptively transferred. Moreover, these TGF-β2 Tregs have increased inducible T-cell costimulatory (ICOS) expression. The ability of TGF-β2 to induce Tregs was reversed when a TGF-β2 neutralizing antibody was used, while this antibody had no effect on TGF-β1 induced Tregs. The work herein describes, for the first time, a novel isoform of TGF-β that may be involved in the generation of Tregs with implications in all areas of immunology.